Location: Children's Nutrition Research Center
2024 Annual Report
Objectives
Objective 1. Characterize oxalate catabolic activity in low and high oxalate plants of dietary importance such as leafy greens harvested at different stages of development.
Subobjective 1A: Characterize dynamic changes in oxalic acid and calcium oxalate crystal formation and assess mineral bioavailability in low and high oxalate leafy plants at different developmental stages
Subobjective 1B: Measure oxalate catabolic activity in low and high oxalate leafy plants at different developmental stages.
Objective 2. Identify and characterize in a model plant system the genes and encoded proteins responsible for each step in a novel pathway of oxalate catabolism.
Subobjective 2A: Isolate and biochemically characterize the putative enzymes responsible for catalyzing the remaining steps in the CoA-dependent pathway of oxalate catabolism
Subobjective 2B: Assignment of each putative enzyme to the CoA-dependent pathway of oxalate catabolism.
Objective 3. Determine the influence of the newly identified oxalate catabolism pathway on the nutritional composition, phytochemical profile, and production characteristics in plants of dietary importance such as leafy greens grown to different stages of maturity (microgreens to mature greens).
Subobjective 3A: Manipulate oxalate catabolism in leafy greens.
Subobjective 3B: Assess the impact of oxalate catabolism on leafy green growth.
Subobjective 3C: Assess the impact of the manipulation of oxalate catabolism on the nutritional quality of leafy greens.
Objective 4: Establish the relationship between genetic background and mineral element bioaccessibility in spinach.
Objective 5: Evaluate carotenoid bioaccessibility as a function of spinach developmental stage.
Approach
Although oxalic acid is known to impact numerous biological processes in a broad range of organisms, our understanding of the mechanisms regulating its turnover are not well understood. This is especially true in plants. To begin to fill these gaps in our knowledge we plan to first assess the oxalate catabolic activity in low and high oxalate plants of dietary importance at different stages of development.The information gained from the assessment would be of use to consumers trying to reduce dietary oxalate loads and scientists interested in gaining new insights into the mechanisms regulating oxalate metabolism in plants. We will also identify and characterize in a model plant system the genes and encoded proteins responsible for each step in the CoA-dependent pathway of oxalate catabolism. The findings obtained will contribute significantly to our understanding of oxalate turnover and will set a foundation for future investigations into oxalate metabolism in a number of organisms ranging from microbes to plants. Additionally, researchers will profile a genetically diverse population of spinach accessions for mineral and carotenoid bioaccessibility using an in vitro digestion approach.
Progress Report
Dietary oxalic acid, consumed primarily through plant-based foods, has been shown to contribute to the formation of kidney stones, decrease the nutritional value of edible plants by acting as an antinutrient, and even exacerbate symptoms associated with other conditions such as autism. The incidence of these negative impacts appears to be rising and will most likely continue to rise with the recommendations for an increase in the consumption of fruits and vegetables. Although dietary oxalic acid has been known to have a negative impact on human health our understanding of the mechanisms regulating its concentration in plant foods is lacking. Recently, we identified an oxalyl-CoA synthetase that is responsible for catalyzing the first step in a previously uncharacterized pathway of oxalate degradation in the plant model Arabidopsis. In this project we extend our initial finding by defining this pathway of oxalate degradation and assessing its influence on the nutritional quality of dietary important plants.
First, we determined whether this novel pathway of oxalate degradation plays a role in regulating the concentration of oxalate found in selected leafy greens. For Objective 1, we completed the evaluation of calcium oxalate crystal formation, oxalate degradation activity, oxalate concentration, mineral analysis, and biomass analysis in different spinach (PI169688, PI648964, PI1335782, and NSL6095) and kale (Premier and Dwarf blue curled vates) varieties at 14, 24, and 44 days after germination which correspond to the micro-, baby-, and mature-leafy greens, respectively, that are marketed to the general public. This information coupled with our previous results can aid the consumer in making informed decisions regarding which stage of spinach to consume since oxalate can inhibit the consumer’s ability to absorb certain minerals, such as calcium, and also contribute to the formation of kidney stones.
Second, we identified and characterized the genes and encoded enzymes responsible for catalyzing each step in this pathway of oxalate catabolism. For Sub-objective 2A we completed cloning, expression, purification, and partial characterization of two putative formyl-coenzyme A hydrolases and formate dehydrogenase, which are enzymes predicted to catalyze the third and fourth step in the pathway of oxalate degradation. There is only one gene encoding formate dehydrogenase in both spinach and Arabidopsis and this enzyme was found to reside in the mitochondria in both plants. In addition, we completed the isolation and characterization of Arabidopsis plants that lack a functional copy of each gene encoding the enzymes for each of the four proposed steps in the pathway of oxalate degradation. Moreover, we completed transcriptomics of the Arabidopsis aae3 mutant. This study provided valuable information regarding the importance of this enzyme and the oxalate degradative pathway in plant growth and development.
Third, we initiated a study to determine the influence of this pathway of oxalate catabolism on the nutritional quality of spinach. Efficient generation of stable transgenic spinach plants with altered oxalate degradative capacity was found to be difficult and inefficient. Thus, as part of Objective 3 we initiated and established a new approach to alter the oxalate degradative capacity of spinach utilizing virus-inducing gene silencing (VIGS) technology. This year, we created and expressed a number of VIGS constructs in spinach. The first set of constructs targeted the different genes of the spinach oxalate degradation pathway. VIGS of Acyl-activating enzyme (AAE) 3 and Oxalate decarboxylase (OXC), genes encoding the first and second steps of the oxalate degradation pathway, respectively, were found to result in plants with an increase in soluble oxalate concentration. The second set of VIGS constructs targeted five putative glycolate oxidase (GLO) genes in spinach. Glycolate oxidase has been suggested to produce oxalate leading to calcium oxalate crystal accumulation. Microscopic analysis of the leaves from the spinach plants expressing the GLO VIGS construct revealed a reduction in calcium oxalate crystals. Oxalate measurements will be conducted to confirm the observed reduction in calcium oxalate. Furthermore, we have created an AAE3 over-expression construct. We are in the process of testing whether expression of this construct results in spinach plants with reduced oxalate. Our hope is that the expression of this construct will result in a reduction in leaf oxalate concentrations.
Iron deficiency is a public health concern in the U.S. and abroad for adolescent girls, people who can become pregnant, and pregnant people. For Sub-objective 4A, we grew 30 distinct varieties of spinach under controlled conditions and harvested plants when they had 6-8 mature leaves. This process was repeated a total of three times. We determined through a collaboration with an ARS researcher in Ithaca, New York, that the bioavailability of iron from spinach was < 1%. With the completion of Sub-objective 4A, we used these data to secure external funding to determine the mechanism of poor bioavailability of iron from spinach. We hypothesized that a class of compounds in spinach called flavonoids are responsible for low iron bioavailability and may also affect iron bioavailability from other foods that are consumed together during a meal. Flavonoid concentrations can be manipulated by different amounts and types of light, so we also developed a secondary aim to explore how different qualities of light influence iron bioavailability – the first study of its kind. This year, we developed a novel method to quantify spinach flavonoids since existing methods only quantify a few at a time. We developed a high throughput extraction and mass spectrometry method to quantify over 40 spinach flavonoids simultaneously allowing us to see how the entire pathway is changing in response to light or other parameters of interest. While the bioavailability studies are ongoing, we determined that a specific combination of light could reduce spinach flavonoids by approximately 50%. If flavonoids do indeed impact iron bioavailability from spinach containing meals, we will likely be able to manipulate iron bioavailability by growing spinach under different wavelengths of light. Additional studies that arose from this Sub-objective include examining the role of genetic diversity on flavonoid bioaccessibility and the role of salinity stress on spinach flavonoid biosynthesis. These studies are underway and will be completed in the coming months.
Sub-objectives 4B and 5A are focused on carotenoids, fat-soluble plant pigments that are associated with positive health benefits and were completed last year. We previously found that microgreens are a poor predictor of carotenoid bioaccessibility (the amount made available for absorption into that body) compared to mature plants and that overall, microgreens are substantially worse at delivering carotenoids than their mature counterparts. We conducted additional experiments in mature spinach plants to explore how other environmental factors such as light quality as well as nutrient and salinity stress can affect carotenoid bioaccessibility. Both carotenoid content, bioaccessibility, and total yield can be modified to varying degrees by different environmental aspects. In particular, we found one salinity treatment that enhanced carotenoid delivery potential and spinach yield by approximately 30%, providing evidence that stress (or molecular pathways related to stress) could be leveraged to generate nutritionally enhanced plant foods. These findings also provide preliminary data for future studies examining how conditions associated with climate change may affect the quality of our food.
To summarize the entire life of this project (April 2022 – current), we have established that spinach contains a family of flavonoids that drastically reduces the absorption of iron, developed methodology to quantify these compounds, and determined that these compounds can be significantly altered by changing the quality and quantity of light during the growing period. We have also explored how genetic diversity influences the concentration and bioaccessibility of these flavonoids as well as how salinity affects their production in spinach leaves. For carotenoids, we identified that carotenoid concentration and bioaccessibility are separate traits and bioaccessibility may be leveraged by breeders in the future. We determined that microgreens are a poor source of carotenoids due to an inability to release these compounds during digestion compared to mature counterparts. Microgreens are also a poor predictor of carotenoid bioaccessibility in mature spinach plants. We generated novel cultural techniques that can be used to alter the delivery of carotenoids and the yield of spinach by using controlled amounts of stress. During the overall life of the project, we have developed a number of novel 3D printed technologies that serve the scientific community by providing low-cost, high utility tools to make laboratories more functional.
Accomplishments
1. Disruption of the plant enzyme AAE3 results in seed germination impairment. Maintaining seed germination is a crucial part of maintaining a stable food supply. As a step toward gaining a better understanding of the biological role of the plant enzyme AAE3 in seed germination, ARS researchers at the Children's Nutrition Research Center in Houston, Texas, compared plants that contained and plants that lacked the AAE3 enzyme. Our studies suggest that a reduction in seed germination in plants lacking AAE3 are attributable to a decrease in secreted substances that coat the seeds, which serves to aid the seed in water absorption as an initial step in the germination process. In addition, the genes encoding the cellular machinery required to make the seed coating were found to have decreased expression in plants lacking AAE3. Measurements of the secreted seed coating and oxalate suggested that the reduced seed germination correlated with the lack of seed coating rather than the elevated amounts of oxalate. It is our hope that a better understanding of seed germination will be useful in the design of strategies to help us better store plant seeds for use by future generations.